TWI835609B - A modified magnetic iron metal organic framework, its preparation method and an electrochemical sensing set - Google Patents

Abstract

The present invention provides a modified magnetic iron metal organic framework, comprising a magnetic iron metal organic framework, a chemical modifier and a capture probe, wherein the magnetic iron metal organic framework is composed of ferric ions, 2-methylimidazole and L-histidine formed metal organic frameworks. The magnetic iron metal organic framework includes an accommodation space, the magnetic nanoparticles are located in the accommodation space of the magnetic iron metal organic framework, and the chemical modifier and the capture probe are covalently connected to the surface of the magnetic iron metal organic framework. In addition, an electrochemical sensing set with a modified magnetic iron metal organic framework combined with an electrochemical sensor is also proposed, and it is confirmed that the problem of insufficient sensitivity of the traditional metal organic framework combined with the electrochemical sensor can be overcome.

Description

Modified magnetic iron-metal organic framework, preparation method thereof and electrochemical sensing kit

The present invention relates to the field of metal-organic frameworks, and in particular to the field of applying metal-organic frameworks to electrochemical sensors for analyzing target biological substances.

Metal-organic framework (MOF) is an organic-inorganic hybrid material with intramolecular pores formed by self-assembly of metal ions and ligands through coordination reaction. It is the current combination of the fields of inorganic materials and organic materials. Representatives, there have been studies on the application of metal-organic frameworks in the fields of material separation, material adsorption, catalysts, medicine and biological sensing, and have related applications and developments.

Generally, metal organic frameworks are used in biological substance detection using electrochemical methods, so they are generally combined with electrochemical sensors to form electrochemical sensors based on metal organic frameworks. The electrochemical sensors are usually three-electrode systems. Including working electrode (WE), auxiliary electrode (CE) and reference electrode (RE), where the working electrode can act as an electron provider or electron receiver as the voltage changes in the electrolyte environment, where The auxiliary electrode plays a role corresponding to the working electrode. When the working electrode acts as an electron provider, the auxiliary electrode acts as As the electron receiver, when the working electrode acts as the electron receiver, the auxiliary electrode acts as the electron provider, and the reference electrode provides a stable relative potential as a reference.

However, there are still many bottlenecks in the application of metal-organic frameworks combined with electrochemical sensors in the field of biosensing. Since metal-organic frameworks are composed of metal ions and ligands, the molecular orbitals and electronic states of metal ions and ligands The overlap is poor, resulting in limited electron transfer, resulting in a low charge movement rate in the metal-organic framework, and the metal-organic framework outside the metal-organic framework is affected by chemical instability, insufficient conductivity, and large particle size, affecting the metal-organic framework. The sensitivity of the skeleton combined with the electrochemical sensor for the analysis of target biological substances has led to the problem of insufficient sensitivity of many electrochemical sensors based on metal organic frameworks.

An object of the present invention is to solve the problem of insufficient sensitivity of electrochemical sensors based on metal organic frameworks.

Therefore, based on one purpose of the present invention, the present invention provides a modified magnetic iron metal organic framework, including: magnetic iron metal organic framework, chemical modifications and capture probes, wherein the magnetic iron metal organic framework is composed of ferric ions, A metal-organic framework formed by coordination combination of 2-methylimidazole and L-histidine, and the magnetic iron-metal organic framework includes an accommodation space, and the magnetic nanoparticles are located in the accommodation space of the magnetic iron-metal organic framework. The chemical modification and the capture probe are covalently connected to the surface of the magnetic iron metal organic framework; the chemical modification is thionine; the capture probe is used to bind to the target biological substance in the specimen, and the capture probe can be an oligo Nucleotides, antibodies or peptide chains.

The specimens include saliva, urine, serum, plasma, whole blood, cells, cell extracts, lymph, cerebrospinal fluid, amniotic fluid, alveolar flush fluid, sweat, tears, sputum, pleural effusion, ascites, synovial fluid, and semen. or feces.

The biological substances are nucleic acids, peptides, proteins or compounds.

The invention provides a method for preparing a modified magnetic iron metal organic framework. The steps include: 1-ethyl-3(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, and magnetic iron metal The organic skeleton and water are mixed and reacted at room temperature (25°C), and then rinsed with pure water. Then, thionine, capture probe and water are added, and the reaction is mixed at room temperature (25°C) to obtain modified Magnetic ferrometal-organic framework.

The present invention provides a method for preparing a modified magnetic iron metal organic framework, which further includes adding thionine, capture probe and water, first rinsing with pure water, and then drying in an oven to obtain a modified magnetic iron metal organic framework. Magnetic ferrometal-organic framework.

Among them, the magnetic iron metal organic framework is composed of magnetic nanoparticles, L-histidine acid, polyvinylpyrrolidone, water and methanol. Then ferric chloride hexahydrate and water are added and left to react at 4°C. Finally, the It is formed by mixing 2-methylimidazole and methanol at room temperature.

The particle size of the magnetic nanoparticles is less than 1,000 nanometer (nm).

The present invention provides a first electrochemical sensing kit, including an electrochemical sensor, a modified magnetic iron metal organic framework as described above, a detection probe and a redox enzyme; wherein the electrochemical sensor includes: The working electrode includes a surface for detecting changes in current, and a back surface opposite the surface. A magnet is provided on the back of the working electrode; a detection probe is used to combine with the target biological substance, and the detection probe is modified with A first label; wherein the redox enzyme is used to perform a redox reaction, and the redox enzyme is modified with a second label, and the second label is used to bind to the first label.

A second electrochemical sensing set, including an electrochemical sensor, a modified magnetic iron-metal organic framework as described above, a capture probe and an embedding agent; wherein the electrochemical sensor is used to perform electrochemistry Analysis, the electrochemical sensor includes a working electrode, the working electrode includes a surface for detecting current changes, and a backside opposite to the surface, and a magnet is provided on the backside of the working electrode; wherein the capture probe is an oligonucleotide; wherein The intercalating agent is used to embed the binding site between the capture probe and the target biological substance and perform a redox reaction; the capture probe is an oligonucleotide; and the target biological substance is a nucleic acid.

The second electrochemical sensing kit further includes a detection probe. The detection probe is used to combine with the target biological substance, and the intercalating agent will be embedded in the combination of the detection probe and the target biological substance, wherein the detection probe is an oligonuclear substance. glycosides.

The electrochemical analysis is differential pulse voltammetry (DPV), cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) or square wave voltammetry (square wave). voltammetry,SWV).

In summary, the present invention is an electrochemical sensor based on a magnetic iron metal organic framework that not only overcomes the problem of insufficient sensitivity of general metal organic framework combined electrochemical sensors, but also has good specificity and a broad range. Quantitative range.

Figure 1 is the spectral diagram analyzed by Fourier transform infrared spectrometer of MNP, skeleton A, skeleton B, skeleton C and skeleton D.

Figure 2 is an image of the crystal of skeleton A taken by a scanning electron microscope.

Figure 3 is an image of the crystal of skeleton B taken by a scanning electron microscope.

Figure 4 is an image of MNP crystals taken by a scanning electron microscope.

Figure 5 is an image of the crystal of skeleton C taken by a scanning electron microscope.

Figure 6 is an image of a crystal of skeleton D taken with a scanning electron microscope.

Figure 7 shows the hysteresis curves of MNP, skeleton C and skeleton D analyzed by superconducting quantum interference instruments.

Figure 8 is a regression analysis of the current intensity generated by target nucleic acid aqueous solutions of different concentrations when the target nucleic acid aqueous solution is tested for wasabi peroxidase using the first electrochemical sensing kit. The measurement specimens are saliva, urine and serum. Figure.

Figure 9 is a regression analysis diagram of the current intensity generated by the target nucleic acid aqueous solution with different concentrations when the target nucleic acid aqueous solution is tested using the second electrochemical sensing kit for intercalating agents and the measurement specimens are saliva, urine and serum.

Figure 10 shows the target virus solution using the first electrochemical sensing kit for wasabi peroxidase testing and the second electrochemical sensing kit for intercalating agent testing. When the sample is serum, the target virus solutions at different concentrations are measured. Regression analysis plot of the current intensity produced.

Figure 11 shows the target extract using the first electrochemical sensing kit for wasabi peroxidase testing and the second electrochemical sensing kit for intercalating agent testing. When the sample is serum, different concentrations of the target extract are measured. Regression analysis plot of the current intensity produced.

Figure 12 is a regression analysis diagram of the current intensity generated by parathyroxine aqueous solutions of different concentrations when the first electrochemical sensing kit was used to test the wasabi peroxidase on the parathyroxine aqueous solution, and the measurement sample was serum.

Figure 13 is a regression analysis diagram of the current intensity generated by S protein solutions of different concentrations when the S protein solution is tested for wasabi peroxidase using the first electrochemical sensing kit, and the measurement sample is serum.

Figure 14 is a regression analysis diagram of the current intensity generated by microRNA solutions of different concentrations when the microRNA solution is tested for intercalation using the second electrochemical sensing kit and the sample is serum.

Although the numerical ranges and parameters used to define the present invention are approximate values, the relevant values in the specific embodiments are presented as accurately as possible. Any numerical value, however, inherently contains the standard deviation resulting from the individual testing methods used. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Alternatively, the word "about" means that the actual value falls within an acceptable standard error of the mean, which is determined by the considerations of a person of ordinary skill in the art to which this invention belongs. Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying patent claims are approximate values and may be changed as required. At a minimum, these numerical parameters should be understood to mean the number of significant digits indicated and the value obtained by applying ordinary rounding.

In various embodiments of the present invention, the solvent in the "aqueous solution" is ultrapure water, for example, ferric chloride hexahydrate aqueous solution, which means that ferric chloride hexahydrate is used as the solute and ultrapure water is used. As the solvent, the rest can be deduced in the same way, but in actual implementation, it is not limited to ultrapure water. Pure water, deionized water, hydrodistilled water or double distilled water can also be used as the solvent.

In various embodiments of the present invention, the solvent in the "methanol solution" is methanol. For example, 2-methylimidazole methanol solution means that 2-methylimidazole is used as the solute and methanol is used as the solvent. Others And so on.

In the following electrochemical analyses, a screen-printed three-electrode (TE-100, Zensor, Taichung) was used as an electrochemical sensor. The screen-printed three-electrode includes a working electrode (WE) and an auxiliary electrode (counter electrode). , CE) and reference electrode (reference electrode (RE)), where the working electrode can act as an electron provider or electron receiver as the voltage changes in the electrolyte environment; the auxiliary electrode plays a role corresponding to the working electrode. When the working electrode When acting as an electron provider, the auxiliary electrode acts as an electron receiver, and when the working electrode acts as an electron receiver, the auxiliary electrode acts as an electron provider; the reference electrode provides a stable relative potential as a reference, and electrochemical analysis includes Differential pulse voltammetry, but the actual implementation is not limited to this, it can also be electrochemical analysis methods such as cyclic voltammetry, electrochemical impedance spectroscopy or square wave voltammetry; among them, cyclic voltammetry and electrochemical impedance In the spectrometry method, the electrolyte solution used is 3mM potassium ferricyanide solution, where the 3mM potassium ferricyanide solution is prepared by adding 98.8 mg of potassium ferricyanide and 10 ml of 0.1M phosphate buffered saline (Phosphate buffered saline, The preparation is completed after mixing PBS); in differential pulse voltammetry and square wave voltammetry, the electrolyte solution used is hydroquinone/hydrogen peroxide solution or 10mM trishydroxymethylaminomethane hydrochloride ( Tris-HCL) aqueous solution; wherein the preparation method of the hydroquinone/hydrogen peroxide solution used in differential pulse voltammetry is to mix a 10mM hydroquinone solution and a 10mM hydrogen peroxide solution at a ratio of 1:1 The preparation is completed by mixing at a volume ratio of Take 100μL of 100mM hydroquinone solution and add 900μL of 0.1M phosphate buffer to obtain a 10mM hydroquinone solution; The 10mM hydrogen peroxide solution is to take 1 μL of 10M hydrogen peroxide solution, and then add 999 μL of 0.1M phosphate buffer to obtain a 10mM hydrogen peroxide solution.

The invention provides a modified magnetic iron metal organic framework, which includes: a magnetic iron metal organic framework, a chemical modification and a capture probe, wherein the magnetic iron metal organic framework is composed of ferric ions, 2-methylimidazole and L- A metal-organic framework is formed by coordination and combination of histidine acid, and the magnetic iron-metal organic framework includes an accommodation space. The magnetic nanoparticles are located in the accommodation space of the magnetic iron-metal organic framework. The chemical modifications and capture probes are Valently connected to the surface of the magnetic iron metal organic framework; wherein the chemical modification is thionine; wherein the capture probe is used to bind to the target biological substance in the specimen, and the capture probe can be an oligonucleotide, antibody or peptide chain .

In one embodiment of the present invention, the specimen is saliva, urine, serum, plasma, whole blood, cells, cell extracts, lymph, cerebrospinal fluid, amniotic fluid, alveolar flushing fluid, sweat, tears, sputum, Commonly used clinical specimens include pleural effusion, ascites, synovial fluid, semen or feces.

In one embodiment of the invention, the biological substance is nucleic acid, peptide, protein or compound.

In one embodiment of the present invention, the particle size of the magnetic nanoparticles is less than 1,000 nanometers.

In order to make it easy for those with ordinary knowledge in the technical field to understand the content of the present invention, the present invention will be further described below in conjunction with the embodiments and drawings. Each embodiment is only used to illustrate the technical features of the present invention. The mentioned content It does not limit the invention.

Preparation Example 1: Preparation method of modified magnetic iron metal organic framework

The step includes adding 1 mL of 0.064 g/mL 1-ethyl-3(3-dimethylaminopropyl)carbodiimide aqueous solution and 1 mL of 0.128 g/mL N-hydroxysuccinimide aqueous solution into a beaker and mixing , to form the first stock solution, then take 1 mL of the first stock solution and 1 mL of the 1 mg/mL magnetic iron metal organic framework aqueous solution into a centrifuge tube, and then shake the reaction with a oscillator for 1 hour at room temperature (25°C). make magnetic iron After the carboxyl groups on the L-histidine acid on the metal-organic framework are activated, the magnetic iron-metal-organic framework is separated using a magnetic stand to remove the supernatant and rinse twice with ultrapure water, and then add 500 μL of After adding 8mM thioacetate aqueous solution and 500μL capture probe aqueous solution, the reaction was carried out with a oscillator for 1 hour at room temperature (25°C), and then the modified magnetic iron-metal organic framework was separated using a magnetic stand and the remaining residues were removed. liquid, and finally rinsed twice with ultrapure water and dried in an oven to obtain a "magnetic iron metal organic framework modified with capture probes and thionine"; in which the sulfur provided in the thionine acetate aqueous solution Coryline is a chemical modification; the magnetic iron-metal organic framework modified with capture probe and thionine refers to the magnetic iron-metal organic framework via 1-ethyl-3 (3-dimethylaminopropyl) carbide The diacetyl aqueous solution reacts with 1 mL of 0.128 g/mL N-hydroxysuccinimide aqueous solution to activate the carboxylic acid of L-histidine on the magnetic iron metal organic framework, thereby forming co-organizers with thionine and the capture probe. The surface of the magnetic ferrometal organic framework is modified with capture probes and thionine through valence linkage, and the magnetic ferrometal organic framework modified with the capture probe and thionine is the modified magnetic ferrometal organic framework of the present invention. Framework; in the following examples, "magnetic iron-metal-organic framework modified with capture probes and thionine" is represented by "skeleton E"; in the following examples, the capture probes are respectively the first capture probe, The second capture probe, the third capture probe and the fourth capture probe are taken as examples, wherein the first capture probe is an oligonucleotide, and the nucleotide sequence of the first capture probe is such as SEQ in the sequence listing. As shown in ID NO: 1, the nucleotide sequence of the first capture probe from 5' to 3' direction is TTTTTTTTTTGCGCGACATTCGGAAGAA, with a total of 28 nucleotides, a molecular weight of 8,559.6 Da, and a melting temperature (Tm) ) is 55.5°C, and when the capture probe is the first capture probe, in "Preparation Method of Magnetic Iron Metal-Organic Framework with Modification"...then add 500 μL of 8mM to the centrifuge tube After the thioacetate aqueous solution and 500 μL of the capture probe aqueous solution..." In the step, the concentration of the capture probe aqueous solution is 0.2 μM; the second capture probe is an antibody and is anti-parathyroxine The antibody of the antibody (Anti-Parathyroid Hormone/PTH Antibody, Mouse Monoclonal, product number: 13192-MM02, SinoBiological), and when the capture probe is the second capture probe, in "Preparation of modified magnetic iron metal organic framework "..." in "Method" In the step of adding 500 μL of 8 mM thionine acetate aqueous solution and 500 μL of the capture probe aqueous solution into the centrifuge tube, the concentration of the capture probe aqueous solution is 500 ng/mL; among them, the third capture probe The antibody is an antibody against the S protein (Spike Protein) of the new coronavirus (COVID-19 S-Protein (S1RBD) ELISA Kit, product number: ELV-COVID19S1-1, RayBiotech), and when the capture probe is For the third capture probe, in "Preparation Method of Modified Magnetic Iron Metal-Organic Framework"...then add 500 μL of 8 mM thionine acetate aqueous solution and 500 μL of capture probe into the centrifuge tube. In the step of "after aqueous solution...", the concentration of the capture probe aqueous solution is 500ng/mL; the fourth capture probe is an oligonucleotide, and the nucleotide sequence of the fourth capture probe is as follows As shown in SEQ ID NO: 2 in the list, the nucleotide sequence of the fourth capture probe is AGTGAATTCTACCAGTGCCATA from 5' to 3' direction, with a total of 22 nucleotides, a molecular weight of 6,927.6 Da, and a melting temperature of ( melting temperature (Tm) is 40.9°C, and the 3' end of the detection probe is modified with a 3'-amino-modified group C7, and when the capture probe is the fourth capture probe, the "magnetic iron with modification" In the step "...then add 500 μL of 8mM thionine acetate aqueous solution and 500 μL of the capture probe aqueous solution..." in the "Preparation Method of Metal Organic Framework", The concentration of the capture probe aqueous solution is 0.2 μM; the first capture probe and the fourth capture probe are both customized and synthesized from MDBio, Inc. and dissolved in pure water. The original concentrations are both 100 μM, and then diluted to 0.2 μM with pure water before use; the above is only an example of a capture probe, and the actual implementation is not limited to this. The corresponding capture probe can be designed according to the target biological substance to be detected, and Capture probes are not limited to oligonucleotides or antibodies, but can also be peptide chains and other aptamers.

Preparation Example 2: Preparation method of magnetic ferrometal organic framework in modified magnetic ferrometal organic framework

The steps include adding 122.4mg of magnetic nanoparticles, 1mL of 0.184M L-histidine acid aqueous solution, 2.75mL of methanol and 0.5g of polyvinylpyrrolidone into the beaker, using ultrasound The crusher performs ultrasonic vibration mixing for 30 minutes to complete the mixing to form a second stock solution. Then take 250 μL of 0.2M ferric chloride hexahydrate aqueous solution and 282 μL of the second stock solution in a centrifuge tube to mix, and then centrifuge Place the tube in the refrigerator for reaction at 4°C for 1 hour. Then add 141 μL of 0.54M 2-methylimidazole methanol solution and 327 μL of methanol into the centrifuge tube, and then shake the centrifuge tube at room temperature (25°C). The centrifuge was shaken and mixed for 2 hours, and then the centrifuge tube was put into a centrifuge and centrifuged at 3,000 rpm for 1 minute. The supernatant was removed and the precipitate was left. The precipitate here is the completed magnetic iron metal organic framework. , then rinse with methanol 3 times, then rinse with acetone once, then add acetone so that the acetone liquid level covers the sediment, and then use an ultrasonic crusher to use ultrasonic vibration to dissolve the sediment in acetone. Finally, a vacuum oven is used for vacuum drying to complete the preparation of the magnetic iron metal organic framework; the magnetic iron metal organic framework refers to the trivalent iron ions provided by ferric chloride hexahydrate as metal ions, and 2-methylimidazole. and L-histidine as a ligand, in which metal ions and ligands undergo a coordination reaction to form a magnetic iron-metal organic framework. The magnetic iron-metal organic framework contains accommodation space, and the magnetic nanoparticles are coated in In the accommodation space; polyvinylpyrrolidone is used as a stabilizer, and the average molecular weight of the polyvinylpyrrolidone used is 10,000 Da; in the following examples, the "magnetic iron metal organic framework" is represented by "skeleton C", so the aforementioned The "modified magnetic iron metal organic framework" can also be expressed as "modified framework C".

Preparation Example 3: Preparation method of magnetic nanoparticles in skeleton C

The steps include placing 1.72g of ferric chloride hexahydrate and 0.403g of ferrous chloride in a beaker, dissolving them with 80 mL of ultrapure water to form an iron ion solution, and then heating the iron ion solution to 80°C. , the molar ratio of ferric chloride hexahydrate and ferrous chloride is about 2:1, then add 6.7mL of 25-30wt% ammonia water and stir and mix at a temperature of 80°C for 1 hour to complete the preliminary magnetic properties Preparation of nanoparticles, wherein the preliminary magnetic nanoparticles are formed by the reaction of iron ion solution and ammonia water. Magnetic ferric iron oxide, then add 20 mL of 0.04165 g/mL sodium citrate aqueous solution, heat it to 90°C, stir and mix at 90°C for 1 hour, and then let it stand at room temperature (25°C). Cool until it is cooled to room temperature (25°C), in which the citrate radicals in the sodium citrate aqueous solution will be bound to the surface of the preliminary magnetic nanoparticles to stabilize the structure, and the preliminary magnetic nanoparticles with citrate radicals bound to the surface, These are the magnetic nanoparticles with a particle size of less than 1,000 nanometers. The magnetic nanoparticles are then separated using a magnetic stand to remove the supernatant, rinsed twice with ultrapure water, and finally dried in an oven. , that is, the preparation of magnetic nanoparticles is completed; in the following examples, "magnetic nanoparticles" will be referred to as "MNP" for short. The magnetic nanoparticles are not limited to those prepared by the method for preparing magnetic nanoparticles provided by the present invention, and can also be other magnetic particles with a particle size less than 1,000 nanometers.

Experimental Example 1: Test the effect of different chemical modifications on the skeleton C on the electron transfer rate constant:

In order to confirm the impact of different chemical modifications on the electron transfer ability of skeleton C, the skeleton C was chemically modified to have chemical modifications on the skeleton C. The chemical modifications were toluidine blue (TBO). , neutral red (NR) and thionine (Thi) to form a skeleton C modified with toluidine blue, a skeleton C modified with neutral red and a skeleton C modified with thionine, wherein there is The preparation method of the aniline blue modified skeleton C and the neutral red modified skeleton C is basically the same as the "preparation method of the modified magnetic iron metal organic framework", the difference is that "...then add it to the centrifuge tube In the step "After 500 μL of 8 mM thionine acetate aqueous solution and 500 μL of the capture probe aqueous solution..." replace "500 μL of 8 mM thionine acetate aqueous solution and 500 μL of the capture probe aqueous solution" with respectively "1mL of 4mM toluidine blue aqueous solution", "1mL of 4mM neutral red aqueous solution" and "1mL of 4mM thionine acetate aqueous solution", so the finally obtained products are respectively skeleton C with toluidine blue modification, The skeleton C with neutral red modification and the skeleton C with thionine modification, and then using 0.1M phosphate buffer as the solvent, respectively combine the skeleton C with toluidine blue modification, the skeleton C with neutral red modification and the skeleton C with sulfur. The cordy modified framework C was diluted to a concentration of 1 mg/mL, and 5 μL of each was added to the surface of the working electrode. Then, 95 μL of 3mM potassium ferricyanide solution was added to the surface of the working electrode as the electrolyte solution, and finally cyclic voltammetry analysis was performed. , and conducted two repeated experiments; the working electrode includes a surface for detecting current changes, and a backside opposite to the surface. A magnet is provided on the backside of the working electrode, and the magnetic force makes the toluidine blue-modified skeleton C, with a medium The sex red-modified framework C and the thionine-modified framework C can be fixed on the surface of the working electrode; cyclic voltammetry analysis is performed at scanning rates of 10, 12, 14 and 16 mV/s respectively and is modified by Lavagnini. The electron transfer rate constant (ks) is obtained by converting the Nicholson equation. The unit of the electron rate constant is "square centimeters per second (cm ² /s)". Since this is a duplicate experiment, the value of the electron transfer rate constant is expressed as "Mean ± standard deviation", the experimental results show that the electron transfer rate constant of skeleton C with toluidine blue modification is 0.001293±0.000249cm ² /s, and the electron transfer rate constant of skeleton C with neutral red modification is 0.00147±0.0000099 cm ² /s, the electron transfer rate constant of skeleton C with thionine modification is 0.002813±0.000377cm ² /s The electron transfer rate constant of skeleton C with thionine modification is greater than the electron transfer rate of skeleton C with toluidine blue modification The constant and the electron transfer rate constant of the skeleton C with neutral red modification represent that the skeleton C with thionine modification has the best electron transfer ability.

In order to carry out subsequent qualitative analysis, Preparation Example 4, Preparation Example 5 and Preparation Example 6 are proposed below to prepare iron-imidazole organic framework, iron-histidine-imidazole organic framework and framework C with thionine modification as qualitative Comparative examples in the analysis.

Preparation Example 4: Preparation method of iron-imidazole organic framework

The steps include adding 250 μL of 0.2M ferric chloride hexahydrate aqueous solution, 375 μL of 0.54M 2-methylimidazole methanol solution and 375 μL of methanol into a centrifuge tube and shaking with a shaker for 2 hours at room temperature (25°C). , then put the centrifuge tube into a centrifuge and centrifuge at 3,000 rpm for 1 minute. Then remove the supernatant and leave the precipitate. The precipitate is the iron-imidazole organic skeleton that completes the synthesis. Next, clean the precipitate first. Rinse the substance with methanol three times, and then rinse it once with acetone to wash away the remaining liquid. Then add acetone to the centrifuge tube and make the acetone liquid level cover the sediment. Then use an ultrasonic crusher to vibrate with ultrasonic waves. Dissolve the precipitate in acetone, and finally dry it in a vacuum oven to complete the preparation of the iron-imidazole organic skeleton, where the iron-imidazole organic skeleton refers to the ferric ions provided by ferric chloride hexahydrate as metal ions. And a metal-organic framework formed by coordination reaction with 2-methylimidazole as a ligand; in the following examples, the "iron-imidazole organic framework" is represented by "framework A".

Preparation Example 5: Preparation method of iron-histidine-imidazole organic framework

The steps include: add 250 μL of 0.2M ferric chloride hexahydrate aqueous solution and 100 μL of 0.184M L-histidine acid aqueous solution into a centrifuge tube, then place it in the refrigerator at 4°C for 1 hour to react to complete the iron-histidine acid solution. Preparation, then add 375 μL of 0.54M 2-methylimidazole methanol solution and 275 μL of methanol to the iron-histidine acid solution, and then shake the reaction with a shaker for 2 hours at room temperature (25°C), and then Place the centrifuge tube into a centrifuge and centrifuge at 3,000 rpm for 1 minute. Remove the supernatant and leave the precipitate. The precipitate is the iron-histidine-imidazole organic skeleton that has been synthesized. The precipitate is then treated with methanol. Rinse 3 times and then rinse once with acetone to wash away the remaining liquid. Then add acetone to the centrifuge tube and make the acetone liquid level cover the sediment. Use an ultrasonic crusher to vibrate the sediment with ultrasonic waves. Dissolve the substance in acetone, and finally use a vacuum oven for vacuum drying to complete the preparation of the iron-histidine acid-imidazole organic framework, where the iron-histidine acid-imidazole organic framework refers to the three-phase iron-histidine acid-imidazole organic framework provided by ferric chloride hexahydrate. Valent iron ions are metal ions, and a metal-organic framework formed by coordination reaction with 2-methylimidazole and L-histidine acid as ligands; in the following examples, "iron-histidine-imidazole organic framework" ” are all represented by “Skeleton B”.

Preparation Example 6: Preparation method for preparing skeleton C with thionine modification

The steps are basically the same as the "Preparation method of modified magnetic iron metal-organic framework", the difference is that "...then add 500 μL of 8mM thionine acetate aqueous solution and 500 μL into the centrifuge tube In the step "After capturing the probe aqueous solution...", "500 μL of 8mM thionine acetate aqueous solution and 500 μL of the capture probe aqueous solution" is changed to "1mL of 4mM thionine acetate aqueous solution", that is "Skeleton C with thionine modification" can be obtained, wherein skeleton C with thionine modification refers to the preparation of skeleton C with 1-ethyl-3(3-dimethylaminopropyl)carbodioxide aqueous solution and 1 mL The reaction of 0.128g/mL N-hydroxysuccinimide aqueous solution activates the carboxylic acid of L-histidine on the skeleton C, and then forms a covalent link with thionine, so that the skeleton C is modified with thionine. ; In the following examples, "skeleton C with thionine modification" is represented by "skeleton D", and skeleton D is the skeleton E that does not include the capture probe. In other words, the skeleton E is the skeleton E that is further modified with a capture probe. Skeleton D.

To facilitate subsequent understanding, the abbreviations mentioned above are summarized here. MNP is "magnetic nanoparticles", skeleton A is "iron-imidazole organic skeleton", skeleton B is "iron-histidine acid-imidazole organic skeleton", and skeleton C is "magnetic ferrometal organic framework", framework D is "skeleton C modified with thionine", and framework E is "magnetic ferrometal organic framework modified with capture probe and thionine".

Qualitative analysis was carried out as follows, which includes dynamic light scattering particle size analyzer and interface potential analyzer (particle size and zeta potential analyzer) analysis, Fourier transform infrared spectrometer (Fouries transform infrared, FITR) analysis, scanning electron microscope ( scanning electron microscope (SEM) analysis and superconducting quantum interference device magnetometer (superconducting quantum interference device magnetometer) analysis.

Experimental Example 2: Dynamic light scattering particle size analyzer and interface potential analyzer analysis

By dissolving each sample in ultrapure water to form a corresponding sample aqueous solution, and using a dynamic light scattering particle size analyzer and an interface potential analyzer to analyze the average particle size of each sample in each sample solution using the light scattering method, the samples include MNP, skeleton A, skeleton B, skeleton C and skeleton D. The numerical presentation method of the average particle size of each sample is "mean ± standard deviation", and the unit of the average particle size is "nanometers". nanometer (nm)", the experimental results show that the average particle size of MNP is 308.0±26.28nm, the average particle size of skeleton A is 253.7±22.49nm, and the average particle size of skeleton B formed by introducing L-histidine acid is 281.6±20.33nm, the average particle diameter of framework C formed by further introducing MNP is 439.2±17.23nm, and the average particle diameter of framework D formed after adding thionine modification is 491.7±42.72nm.

Experimental example 3: Fourier transform infrared spectrometer analysis

Please refer to Figure 1. Fourier transform infrared spectrometer is used to analyze the molecular vibration conditions in each sample to identify each functional group. Each sample is pre-processed by the pressurized tablet method before analysis. Among them, the pressurized tablet method The dispersants used in are all potassium bromide (KBr), and the samples include skeleton A, skeleton B, MNP, skeleton C and skeleton D. The X-axis is the wave number, where the wave number is the reciprocal of the wavelength, so the unit of the X-axis is cm ^-1 , the Y-axis is Transmittance, and the Y-axis unit is %. The experimental results show that the C=C functional group at 1633cm ^-1 and the NH functional group at 3743cm ^-1 of the skeleton B can be observed. , C=NC functional group at 2349cm ^-1 and OH functional group at 3416cm ^-1 , among which the C=NC functional group at 2349cm ^-1 is the functional group characteristic of 2-methylimidazole; MNP has a main structure of tetroxide Fe-O functional group at 585 cm ^-1 can be clearly observed, which is the main functional group characteristic of MNP; skeleton C and skeleton D both contain L-histidine, so it can be observed at 1099 cm ^-1 CH functional group, and because both skeleton C and skeleton D contain MNP, the Fe-O functional group at 585 cm ^-1 can also be observed. At the same time, since skeleton C and skeleton D also contain 2-methylimidazole, so also The C=NC functional group at 2349cm ^-1 can be observed. In addition, since skeleton D is modified with thionine, the signal of the OH functional group at 3416cm ^-1 is more obvious, proving that thionine is successfully modified on the sample.

Experimental Example 4: Scanning Electron Microscope Analysis

Please refer to Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6, which are images of the crystal structure of each sample taken by a scanning electron microscope at the same magnification. The samples include skeleton A, skeleton B, MNP, skeleton C, skeleton D; as shown in Figure 2, the crystal structure of skeleton A presents a nearly cubic structure; as shown in Figure 3, the crystal structure of skeleton B presents an irregular block structure; Figure 4, Figure 5 and 6, the crystals of MNP, the crystals of framework C, and the crystals of framework D all present a particle stacking structure.

Experimental example 5: Superconducting quantum interference element magnetometer analysis

Please refer to Figure 7. Each sample was analyzed using a superconducting quantum interference device magnetometer. The superconducting quantum interference device (Superconducting quantum interference device) applied magnetic fields of different strengths to each sample, causing the magnetism of each sample to change in intensity. , in which a magnetic field intensity range of -10,000 to 10,000 Oersted (Oersted, Oe) is applied to each sample, and the corresponding magnetic changes produced by each sample according to the applied magnetic field intensity are observed, where the unit of magnetism is electromagnetic unit/gram (emu) /g) to obtain the hysteresis curves of each sample, in which the samples include MNP, skeleton C, and skeleton D. Among them, MNP is mainly composed of magnetic ferroferric oxide, so it can be observed that the detected magnetic intensity increases with The magnetic field changes significantly, so MNP is used as the reference standard; the magnetic intensity range of MNP is -60.5827emu/g to 60.64334emu/g, and the magnetic intensity range of skeleton C is -62.0049emu/g to 61.98947emu/ g, the magnetic intensity variation range of skeleton D is -55.8825emu/g to 55.82392emu/g. The magnetic intensity variation range of framework C is similar to that of MNP. The magnetic intensity variation range of framework D is larger than that of MNP. The intensity change range and the magnetic intensity change range of skeleton C are narrow. However, this experiment confirmed that even skeleton D still has obvious magnetic intensity changes as the magnetic field intensity changes. Therefore, it is confirmed that both skeleton C and skeleton D successfully coated magnetic nanoparticles, and It is confirmed that both skeleton C and skeleton D are magnetic.

The experimental results of the aforementioned Experimental Example 2, Experimental Example 3, Experimental Example 4 and Experimental Example 5 prove that there are indeed differences between the skeleton D of the present invention and other comparative examples in terms of average particle size, functional groups, and crystal appearance, and the skeleton D is magnetic, so it can be attracted by a magnetic base or magnet. To separate the skeleton D, the following test is performed on the efficacy of the skeleton D further modified with a capture probe combined with an electrochemical sensor to detect the target biological substance to be detected, that is, the skeleton E combined with an electrochemical biosensor is used The present invention provides the following electrochemical sensing kit for detecting target biological substances to be detected, and subsequently performs wasabi peroxidase testing and intercalating agent testing under various experimental conditions.

The invention provides an electrochemical sensing kit, which includes: an electrochemical sensor, a skeleton E, a detection probe and an oxidation-reduction enzyme. The electrochemical sensor is used for electrochemical analysis, and the electrochemical sensor includes: The working electrode includes a surface for detecting current changes and a back surface opposite the surface. A magnet is provided on the back of the working electrode; the detection probe is used to combine with the target biological substance, and the detection probe is modified with a third A label; wherein the redox enzyme is used to perform a redox reaction, and the redox enzyme is modified with a second label, and the second label is used to bind to the first label; wherein the capture probe can be an oligonucleotide , antibody or peptide chain; wherein the detection probe can be an oligonucleotide, antibody or peptide chain; wherein the target biological substance can be a nucleic acid, peptide, protein or compound; hereinafter referred to as the first electrochemical sensing kit The first electrochemical sensing kit was used in the subsequent experimental examples of wasabi peroxidase testing.

The present invention provides another electrochemical sensing kit, including: an electrochemical sensor, a skeleton E, a detection probe and an embedding agent, wherein the electrochemical sensor is used for electrochemical analysis, and the electrochemical sensor includes: The working electrode includes a surface for detecting changes in current, and a backside opposite to the surface. A magnet is provided on the backside of the working electrode; the detection probe is used to combine with the target biological substance; and the embedding agent is used to embed the capture probe. The binding site between the needle and the target biological substance, and the binding site between the embedded detection probe and the target biological substance, and a redox reaction is performed; the capture probe and the detection probe are both oligonucleotides; and the target biological substance is nucleic acid. ;Hereinafter referred to as this electrochemical sensing kit The second electrochemical sensing set, the second electrochemical sensing set is used in subsequent experimental examples of testing the embedding agent. In an embodiment of the invention, the second electrochemical sensing set may not include a detection probe.

In the following wasabi peroxidase test using the first electrochemical sensing set and the intercalator test using the second electrochemical sensing set, the target biological substance sample used is a sample including the first target biological substance or includes Samples of the second target biological substance, samples including the third target biological substance and samples including the fourth target biological substance are tested, wherein the first target biological substance, the second target biological substance, the third target biological substance and the third target biological substance are tested. The four target biological substances are all examples of target biological substances. The first target biological substance is a partial fragment of the synthetic N-gene of the new coronavirus (SARS-CoV-2). As shown in SEQ ID NO: 3 in the sequence listing, the nucleotide sequence of the first target biological substance from 5' to 3' direction is TTCTTCGGAATGTCGCGCTTTTTTCAATGCTGCAATCGTGCGTCA, with a total of 45 nucleotides, a molecular weight of 13,774.9Da, and melted The temperature is 69.1°C; including samples containing the first target biological substance. The following three modes are provided. The first mode is to customize the synthesis of the first target biological substance from MDBio, Inc., of which the first The target biological substance is dissolved in pure water, and the concentration of the first target biological substance is 100 μM. Hereinafter, the first aspect is called "target nucleic acid aqueous solution"; the second aspect is to the RNAiCore core facility of Academia Sinica The customized recombinant lentivirus carrying the plasmid of the first target biological substance is hereinafter referred to as the "target virus". Before subsequent tests, the target virus will first replicate the target virus through a polymerase chain reaction, so that the target virus The virus reaches the desired replication number; the third method is to further use the viral DNA/RNA extraction kit (Vrial DNA/RNA Kit) of CatchGene Co., Ltd. to extract the viral nucleic acid of the target virus, where the viral nucleic acid is Including the first target biological substance, the extraction method is carried out in accordance with the product operating instructions of the viral DNA/RNA extraction kit to obtain the first target biological substance. The nucleic acid extraction solution of biological substances, hereafter the nucleic acid extraction solution including the first target biological substance will be referred to as the "target extraction solution". The target extraction solution will undergo nucleic acid quantification to confirm the concentration of the target extraction solution before subsequent tests; The second target biological substance is parathyroid hormone, and the sample including the second target biological substance is parathyroid hormone solution (Parathyroid Hormone/PTH Protein, Human, Recombinant (aa 32-65, GST Tag), product number: 13192-H09E , SinoBiological); the third target biological substance is the S protein of the new coronavirus, and the sample including the third target biological substance is the spike protein solution of the new coronavirus (COVID-19 S-Protein (S1RBD) ELISA Kit, item number : ELV-COVID 19S1-1, RayBiotech), hereinafter referred to as the "target protein solution"; the fourth target biological substance is the sequence of a synthetic microRNA (microRNA), of which microRNA-183 is used as an example , the fourth target biological substance is shown as SEQ ID NO: 4 in the sequence listing. The nucleotide sequence of the fourth target biological substance is UUAGGCACUGGUAGAAUUCACU from 5' to 3' direction, with a total of 22 nucleotides, including The sample of the fourth target biological substance is customized to synthesize the fourth target biological substance from MDBio, Inc., in which the fourth target biological substance is dissolved in pure water, and the concentration of the fourth target biological substance is is 100 μM, hereafter referred to as "microRNA solution"; in addition, in order to simulate the state in which the target biological substances to be detected clinically are present in the specimen, the specimen is first centrifuged at 15,000 rpm for 3 minutes to remove impurities. Precipitate, and take the supernatant after centrifugation of the specimen as a diluent, where the diluent is used to dilute the sample including the first target biological substance or the sample including the second target biological substance, and is used to prepare the diluent in the following tests Samples include saliva, urine, and serum, but the actual implementation is not limited to these. In addition to saliva, urine, and serum, samples can also be plasma, whole blood, cells, cell extracts, lymph, cerebrospinal fluid, Amniotic fluid, alveolar flushing fluid, sweat, tears, sputum, pleural effusion, ascites, synovial fluid, semen or feces and other samples used clinically.

Method for wasabi peroxidase testing using the First Electrochemical Sensing Kit: The steps include preparing each test sample, wherein the method of preparing each test sample is to dilute the target biological material sample with a diluent to complete the preparation of each test sample; and then soak the skeleton E in a 3wt% bovine serum albumin aqueous solution. Shake and mix with a oscillator for 10 minutes to block the skeleton E, in which 3wt% bovine serum albumin aqueous solution is the blocking buffer; then separate the blocked skeleton E with a magnetic stand and wash it 5 times with 0.1M phosphate buffer. , to remove the residual blocking buffer; then use 0.1M phosphate buffer to complete the washing of the scaffold E to a concentration of 1 mg/mL to form a 1 mg/mL scaffold E solution; then add 20 μL of 1 mg/mL The skeleton E solution was added to 20 μL of each test sample, and the mixture was mixed and reacted with a oscillator for 15 minutes, so that the target biological substances in each test sample were captured by the capture probe on the skeleton E; then the magnetic base was used to separate each sample. Detect the skeleton E in the sample and wash it three times with 0.1M phosphate buffer to remove the residual liquid; then add each skeleton E that has reacted with each test sample to the detection probe aqueous solution and let it stand for reaction. The detection probe is combined with the target biological substance captured by the capture probe on the skeleton E; then the magnetic base is used to separate each skeleton E that has reacted with the detection probe aqueous solution, and is first treated with 0.1wt% sodium dodecyl sulfate. Rinse once with aqueous solution and then rinse twice with 0.1M phosphate buffer to remove residual liquid. After using 0.1wt% sodium dodecyl sulfate aqueous solution in this step, rinse with 0.1M phosphate buffer. Buffer flushing, compared with only flushing with sodium dodecyl sulfate aqueous solution, or only flushing with 0.1M phosphate buffer, can better flush out the detection probes that have not reacted with the skeleton E, thereby reducing the subsequent current. The background value signal interferes with the intensity detection; then, each washed skeleton E is added with redox enzyme diluted in 0.1M phosphate buffer for reaction, so that the second label on the redox enzyme is bound to Detect the first label of the probe; then use a magnetic base to separate each skeleton E that has completed the reaction with the redox enzyme, and wash it 5 times with 0.1M phosphate buffer to remove the remaining liquid; then complete the washing Add 20 μL of 0.1M phosphate buffer to each scaffold E to prepare a concentration of 1 mg/mL, and then add 5 μL of to the surface of the working electrode of the electrochemical sensor, and the magnet provided on the back of the working electrode will fix the skeleton E to the surface of the working electrode through magnetic force; then add 95 μL of electrolyte solution to the surface of the working electrode, and finally perform electrochemistry Analysis, in which the electrochemical analysis uses differential pulse voltammetry for analysis to measure the current intensity, and perform three repeated experiments, so the value of the current intensity is expressed as "mean ± standard deviation" based on the results of the three repeated experiments; the electrolyte solution is Hydroquinone/hydrogen peroxide solution; the electrochemical sensor, skeleton E, detection probe and redox enzyme are all provided by the first electrochemical sensing kit; the parameters used in differential pulse voltammetry analysis Medium, the amplitude is 75mV, the voltage range is 200mV to -400mV, the pulse period is 125ms, the pulse width is 115ms, and the increment parameter is 20mV; the oxidoreductase is Streptavidin conjugated wasabi peroxidase (Streptavidin conjugated). Horseradish peroxidase, SHRP, product number: DY998, R&D, USA Florida) is used as an example, in which streptavidin is the second marker, and "...then each washed skeleton E, Add redox enzyme diluted with 0.1M phosphate buffer to react, so that the second label on the redox enzyme binds to the first label on the detection probe..." , the wasabi peroxidase labeled with streptavidin was diluted 25 times or 200 times with 0.1M phosphate buffer, and the reaction time was 10 minutes or 15 minutes; in the following examples, The detection probes take the first detection probe, the second detection probe, and the third detection probe as examples respectively. The first detection probe is an oligonucleotide, and the nucleotide sequence of the detection probe is as shown in the sequence list. As shown in SEQ ID NO: 5, the nucleotide sequence of the detection probe is TGTAGCACGATTGCAGCATTG from 5' to 3' direction, with a total of 21 nucleotides, a molecular weight of 6,886.7Da, and a melting temperature of 52.4°C. And the 5' end of the detection probe is modified with a first label, wherein the first label is biotin, and when the detection probe is the first detection probe, in "using the first electrochemical sensing "Method of Kit for Wasabi Peroxidase Test"...Then add each skeleton E that has reacted with each test sample to the detection probe aqueous solution for static In the step of "setting up a reaction to combine the detection probe with the target biological material captured by the capture probe on the skeleton E...", the volume of the detection probe aqueous solution added is 20 μL, and the concentration of the detection probe is 0.25 μM, and the standing reaction time is 15 minutes. The first detection probe is customized and synthesized from MDBio, Inc., and the first detection probe is dissolved in pure water, and the concentration is 100μM, dilute it with pure water to 0.25μM before use; the second detection probe is an anti-parathyroid hormone antibody modified with the first marker (Anti-Parathyroid Hormone/PTH Antibody, Rabbit Polyclonal, product number: 13192- T08, SinoBiological), in which the first marker is biotin, and when the detection probe is the second detection probe, in "Method for Wasabi Peroxidase Test Using the First Electrochemical Sensing Kit" ...Then, each framework E that has reacted with each detection sample is added to the detection probe aqueous solution and left to react, so that the detection probe combines with the target biological substance captured by the capture probe on the framework E... ..." step, the volume of the added detection probe aqueous solution is 20 μL, and the concentration of the detection probe is 500ng/mL, and the standing reaction time is 10 minutes; the third detection probe is modified with the first The secondary antibody of the marker (Anti-Parathyroid Hormone/PTH Antibody, Rabbit Polyclonal, product number: 13192-T08, SinoBiological), where the first marker is biotin, and when the detection probe is the third detection probe, In the "Method for testing wasabi peroxidase using the first electrochemical sensing kit", "...then add each skeleton E that has reacted with each test sample to the detection probe aqueous solution and let it stand for reaction , the detection probe is combined with the target biological substance captured by the capture probe on the skeleton E..." In the step, the volume of the detection probe aqueous solution added is 20 μL, and the concentration of the detection probe is 500ng /mL, and the standing reaction time is 10 minutes; the above is only an example of a detection probe, and the actual implementation is not limited to this. The corresponding detection probe can be designed according to the biological substance to be detected and the type of capture probe used. needle, and the detection probe is not limited to oligonucleotides or antibodies, but can also be aptamers such as peptide chains.

Method for intercalator testing using a second electrochemical sensing set: The steps include preparing each test sample, wherein the method of preparing each test sample is to dilute the target biological material sample with a diluent to complete the preparation of each test sample; and then soak the skeleton E in a 3wt% bovine serum albumin aqueous solution. Shake and mix with a oscillator for 10 minutes to block the skeleton E, in which 3wt% bovine serum albumin aqueous solution is the blocking buffer; then separate the blocked skeleton E with a magnetic stand and wash it 5 times with 0.1M phosphate buffer. , to remove the residual blocking buffer; then use 0.1M phosphate buffer to complete the washing of the scaffold E to a concentration of 1 mg/mL to form a 1 mg/mL scaffold E solution; then add 20 μL of 1 mg/mL The skeleton E solution was added to 10 μL of each detection sample, and after adding the detection probe aqueous solution and the intercalating agent, use a oscillator to mix and react for 10 minutes, so that the target biological substances in each detection sample were captured by the capture probe on the skeleton E. Captured, and the detection probe is combined with the target biological substance captured by the capture probe on the skeleton E, and the intercalator will be embedded in the hybridization point of the capture probe and the target biological substance, and the detection probe and the target biological substance will be embedded The hybridization place; then use a magnetic base to separate each framework E that has reacted with each detection sample, detection probe aqueous solution and intercalating agent, and first wash it once with 0.1wt% sodium dodecyl sulfate aqueous solution, and then rinse it with 0.1 Rinse twice with M phosphate buffer to remove residual liquid. In this step, after using 0.1wt% sodium dodecyl sulfate aqueous solution, rinse with 0.1M phosphate buffer. Compared with only Using sodium dodecyl sulfate aqueous solution or only 0.1M phosphate buffer can rinse the detection probes that have not reacted with the framework E, thereby reducing the background signal during subsequent current intensity detection. Interference; then add 20 μL of 0.1M phosphate buffer to each skeleton E that has been rinsed to prepare a concentration of 1 mg/mL, and then add 5 μL to the surface of the working electrode of the electrochemical sensor, and the working electrode The magnet set on the back will fix the skeleton E on the surface of the working electrode through magnetic force; then add 95 μL of electrolyte solution to the surface of the working electrode, and finally perform electrochemical analysis. The electrochemical analysis uses differential pulse voltammetry to measure the current. The intensity was measured and three replicate experiments were performed, so the value of the current intensity was based on the three replicate experiments. The test results are expressed as "mean ± standard deviation"; the electrolyte solution is 10mM trishydroxymethylaminomethane hydrochloride aqueous solution; the electrochemical sensor, skeleton E, detection probe and embedding agent are all second electrochemical Provided by the scientific sensing kit; among the parameters used in differential pulse voltammetry analysis, the amplitude is 75mV, the voltage range is 0mV to -800mV, the pulse period is 125ms, the pulse width is 115ms and the incremental parameter is 20mV; which is embedded The agent uses neutral red as an example, and in "...then add 20 μL of 1 mg/mL framework E solution to 10 μL of each detection sample, and add the detection probe aqueous solution and embedded After reagent, use a oscillator to mix and react for 10 minutes, so that the target biological substances in each detection sample are captured by the capture probe on the skeleton E, and the detection probe and the target organism captured by the capture probe on the skeleton E are Substances are combined, and the intercalating agent will embed into the hybridization point between the capture probe and the target biological material, and embed into the hybridization point between the detection probe and the target biological material..." In the step of adding 5 μL of 4mM neutral Red; in the following examples, the detection probe is the first detection probe described in the aforementioned "Steps of Using the First Electrochemical Sensing Kit to Test Wasabi Peroxidase" as an example, and in "Using the First Electrochemical Sensing Kit" "Method of 2 Electrochemical Sensing Kits for Intercalant Testing"... Then add 20 μL of 1 mg/mL framework E solution to 10 μL of each test sample, and add the detection probe aqueous solution and After inserting the intercalating agent, use a oscillator to mix and react for 10 minutes, so that the target biological substances in each detection sample are captured by the capture probe on the skeleton E, and the detection probe and the target captured by the capture probe on the skeleton E are Biological substances are combined, and the intercalating agent will be embedded at the hybridization point between the capture probe and the target biological substance, and the detection probe will be embedded into the hybridization point between the detection probe and the target biological substance..." In the step of adding the detection probe The volume of the aqueous solution is 5 μL, and the concentration of the detection probe aqueous solution is 1 μM; the above is only an example of the detection probe, and the actual implementation is not limited to this. It can be designed according to the biological substances to be detected and the type of capture probe used. Corresponding detection probe.

Experimental Example 6: Using the first electrochemical sensing kit on a target nucleic acid aqueous solution to test wasabi peroxidase

Please refer to Table 1 and Figure 8. In Experimental Example 6, the first electrochemical sensing kit was used to perform a wasabi peroxidase test on the target nucleic acid aqueous solution. For the steps, please refer to "Using the First Electrochemical Sensing Kit to Perform Wasabi Peroxidase" Enzyme test method", in which the target biological substance sample used is a sample including the first target biological substance, wherein the sample including the first target biological substance is a target nucleic acid aqueous solution; the specimens are saliva, urine and serum respectively, and Take the supernatant after centrifugation of each specimen as a diluent; the capture probe on the skeleton E is the first capture probe, because the first capture probe has a nucleotide sequence complementary to a part of the first target biological material. Nucleotide sequence, so the first capture probe on the backbone E can capture the first target biological substance through hybridization; where "the target biological substance sample is diluted with a diluent to complete the preparation of each detection sample" In the step, the target nucleic acid aqueous solution is serially diluted so that the concentrations of the target nucleic acid aqueous solution are 0.005fM, 0.05fM, 0.5fM, 5fM, 50fM, 500fM, 5,000fM and 50,000fM respectively as each detection sample; wherein the detection probe It is a first detection probe. The first detection probe includes a nucleotide sequence that is complementary to another part of the nucleotide sequence of the first target biological substance. Therefore, the first detection probe can hybridize with the first target biological substance. Substances bind, and since the 5' end of the first detection probe is modified with biotin, the streptavidin on the wasabi peroxidase labeled with streptavidin will bind to biotin, so when After the first capture probe on the skeleton E captures the first target biological substance, the first detection probe will bind to the first target biological substance captured by the skeleton E, and the biotin on the first detection probe will be labeled with Streptavidin binds to the streptavidin on wasabi peroxidase, and the wasabi peroxidase in streptavidin-labeled wasabi peroxidase undergoes a redox reaction, so in electrochemical analysis The current change generated during the redox reaction can be detected, and the higher the concentration of wasabi peroxidase labeled with streptavidin, the greater the current intensity; among them, "... Then, each washed scaffold E is added with redox enzyme diluted in 0.1M phosphate buffer for reaction, so that the second label on the redox enzyme binds to the first label on the detection probe. ....." In the step, the wasabi peroxidase labeled with streptavidin was diluted 25 times with 0.1M phosphate buffer, and the reaction time was 15 minutes; the experiment of Experimental Example 6 The results are shown in Table 1 and Figure 8. Table 1 shows the current intensity measured under various concentrations of target nucleic acid aqueous solutions when the samples used were saliva, urine and serum. The concentration unit of the target nucleic acid aqueous solution is fM. , where the unit of current intensity is μA, and is divided into groups according to the types of specimens used, namely saliva, serum and urine; in addition, according to the experimental data in Table 1, the specimens are divided into groups of saliva, urine and serum. Carry out regression analysis and obtain the regression equation of the regression line through regression analysis as follows. The regression equation where the specimen is saliva is expressed as: I=0.4217×ln(x)+7.8546. The determination coefficient of the regression equation is 0.9548 and the slope is 0.4217. μA/fM, the standard deviation of the lowest detection concentration is 0.1340μA, and the calculated limit of detection (LOD) value is 0.9533fM; the regression equation where the sample is urine is expressed as: I=0.3059×ln( x)+9.4438, the determination coefficient of the regression equation is 0.9873, the slope is 0.3059μA/fM, the standard deviation of the lowest detection concentration is 0.0391μA, and the calculated detection limit value is 0.3067fM; where the sample is serum, the regression equation represents is: I=0.445×ln(x)+7.0396, the determination coefficient of the regression equation is 0.9873, the slope is 0.445μA/fM, the standard deviation of the lowest detection concentration is 0.1115μA, and the calculated detection limit value is 0.4511fM; In each regression equation, I represents the current intensity in μA; ln(x) represents the concentration of the target nucleic acid aqueous solution in fM; the slope refers to the slope of the regression line in μA/fM; the minimum detection concentration is The standard deviation refers to the standard deviation calculated from the current intensity values obtained from the results of three repeated experiments in a group with a target nucleic acid aqueous solution concentration of 0.005fM, the unit is μA; the unit of the detection limit value is fM, where the detection limit value is The detection limit is calculated by dividing three times the standard deviation of the lowest detected concentration signal by the slope of the regression line. The detection limit in the following embodiments is calculated in this manner. The calculation formula of detection limit value is as follows:

Figure 8 is a regression analysis graph drawn based on the experimental data in Table 1 and the results of regression analysis on the groups of saliva, urine and serum as specimens according to the experimental data in Table 1; the X-axis in Figure 8 is the concentration of the target nucleic acid aqueous solution, the X-axis unit is fM, the Y-axis is the measured current intensity, the Y-axis unit is μA, and they are grouped according to the type of specimen used, namely saliva, serum and urine.

Experimental Example 7: Using the second electrochemical sensing set to test the intercalating agent on the target nucleic acid aqueous solution

Please refer to Table 2 and Figure 9. In Experimental Example 7, the target nucleic acid aqueous solution was tested using the second electrochemical sensing kit for the intercalating agent. For the steps, please refer to "Method for testing the intercalating agent using the second electrochemical sensing kit." ”, the target biological substance sample used is a sample including the first target biological substance, and the sample including the first target biological substance is a target nucleic acid aqueous solution; the samples are saliva, urine and serum, and each sample is taken The supernatant after centrifugation was used as diluent; among them, the The capture probe is the first capture probe; in the step of "diluting the target biological material sample with each diluent to complete the preparation of each detection sample", the target nucleic acid aqueous solution is serially diluted so that the target nucleic acid aqueous solution The concentrations are 0.005fM, 0.05fM, 0.5fM, 5fM, 50fM, 500fM, 5,000fM and 50,000fM respectively as each detection sample; the detection probe is the first detection probe. Since the intercalating agent will be embedded in the nucleic acid hybridization site, Therefore, the intercalating agent will be embedded in the hybridization site of the first capture probe and the first target biological substance, and will be embedded in the hybridization site of the first detection probe and the first target biological substance, and the intercalating agent will undergo an oxidation-reduction reaction, so it is suitable for electrochemistry. During the analysis, the current change generated during the redox reaction can be detected, and as the concentration of the labeled intercalator is higher, the current intensity will be greater; the experimental results of Experimental Example 7 are shown in Table 2 and Figure 9. Table 2 shows the current intensity measured in target nucleic acid aqueous solutions of various concentrations when the specimens used are saliva, urine and serum. The concentration unit of the target nucleic acid aqueous solution is fM, and the unit of current intensity is μA. According to The types of specimens used are divided into groups, namely saliva, serum and urine. In addition, according to the experimental data in Table 2, regression analysis is performed on the groups where the specimens are saliva, urine and serum. The regression line is obtained through regression analysis. The regression equation is as follows. The regression equation where the specimen is saliva is expressed as: I=0.1131×ln(x)+6.9965. The determination coefficient of the regression equation is 0.9581, the slope is 0.1131μA/fM, and the standard deviation of the lowest detection concentration is 0.1457μA, and the calculated detection limit is 3.8647fM; the regression equation where the sample is urine is expressed as I=0.1844×ln(x)+7.4896, the determination coefficient of the regression equation is 0.9879, and the slope is 0.1844μA/fM , the standard deviation of the lowest detection concentration is 0.1709μA, and the detection limit value is calculated to be 2.7804fM; the regression equation where the sample is serum is expressed as: I=0.3942×ln(x)+7.1625, and the determination coefficient of the regression equation is 0.977, the slope is 0.3942μA/fM, the standard deviation of the lowest detection concentration is 0.0252μA, and the calculated detection limit is 0.1918fM; in each regression equation, I represents the current intensity in μA; where ln(x ) represents the target core The concentration of the acid aqueous solution, in fM; where the slope refers to the slope of the regression line, in μA/fM; where the standard deviation of the lowest detection concentration refers to the group in which the concentration of the target nucleic acid aqueous solution is 0.005fM, from three repeated experiments The standard deviation obtained by calculating the current intensity value obtained as a result is in μA; the unit of the detection limit value is fM. Figure 9 is a regression analysis graph drawn based on the experimental data in Table 2 and the results of regression analysis on the groups of saliva, urine and serum as specimens according to the experimental data in Table 2; the X-axis in Figure 9 is the concentration of the target nucleic acid aqueous solution, the X-axis unit is fM, the Y-axis is the measured current intensity, the Y-axis unit is μA, and they are grouped according to the type of specimen used, namely saliva, serum and urine.

Through the experimental results of Experimental Example 6 and Experimental Example 7, it is confirmed that the first electrochemical sensing kit and the second electrochemical sensing kit provided by the present invention not only have high sensitivity in detecting the first target biological substance, And as the concentration of the added target nucleic acid solution becomes higher, the intensity of the current generated becomes greater, and the concentration of the target nucleic acid solution can be calculated from the detected current through the regression equation, thereby achieving a quantitative effect and being quantifiable. Concentration range is 0.005fM to 50,000fM.

The present invention further tests whether non-target biological substances will be detected when using the first electrochemical sensing set to perform the wasabi peroxidase test and using the second electrochemical sensing set to perform the intercalating agent test. Therefore, the first target biological substance is used as an example of the target biological substance, and a six-base mismatch sequence, a three-base mismatch sequence, a single-base mismatch sequence, and a random sequence are used as non-target biological substances. An example of a substance; the nucleotide sequence of the six-base mismatch sequence is shown in SEQ ID NO: 6 in the sequence listing, and the nucleotide sequence of the six-base mismatch sequence is from 5' to 3' direction. The order is TTCTTCGGAATGTCGCGCTTTTTTTGGCATTGCAATCGTGCTACA, with a total of 45 nucleotides, a molecular weight of 13,790 Da and a melting temperature of 68.2°C, of which the 26th to 31st nucleotides are nucleotides that are different from the first target biological substance; where The nucleotide sequence of the three-base mismatch sequence is shown as SEQ ID NO: 7 in the sequence listing. The nucleotide sequence of the three-base mismatch sequence from 5' to 3' direction is TTCTTCGGAATGTCGCGCTTTTTTTGGTGCTGCAATCGTGCTACA, totaling It has 45 nucleotides, a molecular weight of 13,805 Da, and a melting temperature of 69.1°C. The 25th to 27th nucleotides are nucleotides that are different from the first target biological substance; among them, there is a single base mismatch. The nucleotide sequence of the sequence is shown in SEQ ID NO: 8 in the sequence listing. The nucleotide sequence of the single-base mismatch sequence from 5' to 3' direction is TTCTTCGGAATGTCGCGCTTTTTTCAATGCTGCAATCGTGCTGCA, with a total of 45 nucleotides. , with a molecular weight of 13,775 Da and a melting temperature of 69.0°C, in which the 43rd nucleotide is a nucleotide that is different from the first target biological substance; the random sequence of nucleotide sequences is such as SEQ ID NO in the sequence listing :9, the nucleotide sequence of the random sequence is AAGTAATACGACTCACTATAGGGATGGTAGCGTGAGCAAGG from 5' to 3' direction, with a total of 41 nucleotides, a molecular weight of 13,060 Da and a melting temperature of 68.4°C; six bases are mismatched. The sequence, three-base mismatch sequence, single-base mismatch sequence and random sequence sequence are all customized and synthesized from MD Bio, Inc., and are all dissolved in pure water, and the concentration All are 100 μM, so 100 μM six-base mismatch sequence aqueous solution, 100 μM three-base mismatch sequence aqueous solution, 100 μM single-base mismatch sequence aqueous solution, 100 μM random sequence aqueous solution, and 100 μM six-base aqueous solution were obtained. base error Coordination sequence aqueous solution.

Experimental Example 8: Testing wasabi peroxidase on target biological substances and non-target biological substances using the first electrochemical sensing kit

Please refer to Table 3. In Experimental Example 8, the first electrochemical sensing kit was used to conduct a wasabi peroxidase test on target biological substances and non-target biological substances. The steps are the same as "Using the First Electrochemical Sensing Kit to Test Wasabi Peroxidase". The method of oxidase test is basically the same, in which the specimens are serum, and the supernatant after centrifugation of the specimen is taken as the diluent; the capture probe on the skeleton E is the first capture probe; the detection probe is The first detection probe; wherein "...then add oxidoreductase diluted with 0.1M phosphate buffer to each of the washed scaffolds E for reaction, so that the third oxidoreductase on the oxidoreductase In the step of "binding the second labeling substance to the first labeling substance on the detection probe...", wasabi peroxidase labeled with streptavidin protein was subjected to 25 times in 0.1M phosphate buffer. dilute, and the reaction time is 15 minutes; the difference between Experimental Example 8 and "Method for testing wasabi peroxidase using the first electrochemical sensing kit" is that "... use diluent to test the target biological material sample Dilute to complete the preparation of each test sample..." In the step, use diluent to dilute the target nucleic acid aqueous solution, the six-base mismatch sequence aqueous solution, the three-base mismatch sequence aqueous solution, and the single-base mismatch sequence aqueous solution. The base mismatch sequence aqueous solution and the random sequence aqueous solution were diluted to 50,000 fM as each detection sample, and further included a blank control group. The blank control group was a control group that only used the diluent as the detection sample; experimental results of Experimental Example 8 As shown in Table 3, Table 3 shows the current intensity measured in each test sample when the sample used is serum. The solute contained in the test sample is used as a group. Therefore, the group includes the first target biological substance, six bases Mismatch sequence, three-base mismatch sequence, single-base mismatch sequence and random sequence, in addition to a blank control group; the unit of current intensity is μA; the experimental results of Experimental Example 8 show that the test sample includes The current intensity of the first target biological substance is significantly greater than the current intensity of the test sample including other non-target biological substances and the blank Current intensity in the control group.

Experimental Example 9: Effects of using the second electrochemical sensing set on intercalator testing on target biological substances and non-target biological substances.

Please refer to Table 4. In Experimental Example 9, the target biological substance, that is, the non-target biological substance, was tested using the second electrochemical sensing set. The method is basically the same, in which the specimens are serum, and the supernatant after centrifugation of the specimen is taken as the diluent; the capture probe on the skeleton E is the first capture probe; the detection probe is the first detection probe Needle; The difference between Experimental Example 9 and "Method of using the second electrochemical sensing kit for intercalator testing" is that "...the target biological substance sample is diluted with a diluent to complete the preparation of each test sample ..." step, use diluents to dilute the target nucleic acid aqueous solution, the six-base mismatch sequence aqueous solution, the three-base mismatch sequence aqueous solution, the single-base mismatch sequence aqueous solution and the random sequence. The aqueous solution was diluted to 50,000fM to serve as each test sample, and further included a blank control group. The blank control group was a control group that only used the diluent as the test sample; the experimental results of Experimental Example 9 are shown in Table 4. Table 4 shows the use The sample is serum, and the current intensity measured in each test sample is grouped by the target biological substance or non-target biological substance included in the test sample, so the grouping Including the first target biological substance, six-base mismatch sequence, three-base mismatch sequence, single-base mismatch sequence and random sequence, and also includes a blank control group; the unit of current intensity is μA; The experimental results of Experimental Example 9 show that the current intensity of the target nucleic acid aqueous solution is significantly greater than the current intensity of other non-target biological substances and the current intensity of the blank control group, further verifying that the skeleton E combined with the electrochemical sensor provided by the present invention has good specificity .

Through the experimental results of Experimental Example 8 and the experimental results of Experimental Example 9, it is confirmed that both the first electrochemical sensing kit and the first electrochemical sensing kit provided by the present invention have good specificity and can distinguish the first target. Biological substances and other non-target biological substances.

Experimental Example 10: Using the first electrochemical sensing set to test the target virus solution for wasabi peroxidase, and using the second electrochemical sensing set to test the target virus solution for the intercalator

，最低偵測濃度的標準 差為0.3037μA，計算出偵測極限值為2.8017複製數/μL；其中於嵌入劑測試的回歸方程式為I=0.3372×ln(x)+3.6967，回歸方程式的決斷係數為0.8884，斜率為 0.3372

，最低偵測濃度的標準差為0.2554μA，計算出偵測極限值為2.2 722複製數/μL；於各個回歸方程式中，其中I代表電流強度，單位為μA；其中ln(x)代表目標病毒溶液的濃度，單位為複製數/μL；斜率指回歸直線的斜率，單 位為

；其中最低偵測濃度的標準差指於目標病毒溶液的濃度為0.005複 製數/μL的組別中，由三重複實驗結果獲得的電流強度值計算獲得的標準差，單位為μA；其中偵測極限值的單位為複製數/μL。圖10為根據表5的實驗數據，及根據表5的實驗數據分別對使用的測試方法為山葵過氧化酶測試及嵌入劑測試的組別進行回歸分析的結果，所繪成的回歸分析圖；圖10中X軸為目標病毒溶液的濃度，X軸單位為複製數/μL，Y軸為測得的電流強度，Y軸單位為μA，並且根據使用的測試方法為山葵過氧化酶測試及嵌入劑測試。 Please refer to Table 5 and Figure 10. In Experimental Example 10, the first electrochemical sensing kit was used to perform a wasabi peroxidase test on the target virus. For the steps, please refer to "Using the First Electrochemical Sensing Kit to Test Wasabi Peroxidase.""Method", the target biological substance sample used is a sample including the first target biological substance, the sample including the first target biological substance is the target virus, the sample is serum, and the supernatant after centrifugation of the sample is taken As a diluent, the capture probe on the skeleton E is the first capture probe. In the step of "diluting the target biological substance sample with the diluent to complete the preparation of each detection sample", the target virus is first Perform polymerase chain reaction for amplification and then perform serial dilutions with diluents to form target virus solutions, with the concentrations of the target virus solutions being 6.5 copy number/μL, 65 copy number/μL, 650 copy number/μL, and 6,500. Copy number/μL, 65,000 copy number/μL, 650,000 copy number/μL and 6,500,000 copy number/μL are used as each detection sample; where the detection probe is the first detection probe, where the detection probe is the first detection probe; where "...then add redox enzyme diluted with 0.1M phosphate buffer to each washed scaffold E to react, so that the second label on the redox enzyme binds to the detection probe. In the step of "The first label on the needle...", wasabi peroxidase labeled with streptavidin was diluted 25 times with 0.1M phosphate buffer, and the reaction time was 15 minutes; in addition, in Experimental Example 10, the target virus was tested using the second electrochemical sensing kit for the intercalating agent. For the steps, please refer to "Method for testing the intercalating agent using the second electrochemical sensing kit", in which the The target biological substance sample is a sample including the first target biological substance, wherein the sample including the first target biological substance is the target virus, wherein the sample is serum, and the supernatant after centrifugation of each sample is taken as a diluent, wherein the skeleton The capture probe on E is the first capture probe, in which in the step of "diluting the target biological material sample with a diluent to complete the preparation of each detection sample", the target virus is serially diluted to form the target virus. solution, and the concentrations of the target virus are 6.5 copy number/μL, 65 copy number/μL, 650 copy number/μL, 6,500 copy number/μL, 65,000 copy number/μL, 650,000 copy number/μL and 6,500,000 copy number/ μL is used as each detection sample, in which the detection probe is the first detection probe; the experimental results of Experimental Example 10 are shown in Table 5 and Figure 10. Table 5 shows the target virus at various concentrations when the samples used are serum. The current intensity measured by the solution, where the concentration unit of the target virus solution is copy number/μL, where the unit of current intensity is μA, and is grouped according to the test method used, which are wasabi peroxidase test and intercalator test; in addition According to the experimental data in Table 5, regression analysis was performed on the groups using the wasabi peroxidase test and the intercalating agent test. The regression equation of the regression line obtained through regression analysis is as follows, among which in the wasabi peroxidase test The regression equation of is I=0.3252×ln(x)+5.6362, the determination coefficient of the regression equation is 0.9653, and the slope is 0.3252

, the standard deviation of the lowest detection concentration is 0.3037μA, and the calculated detection limit is 2.8017 copies/μL; the regression equation for the intercalator test is I=0.3372×ln(x)+3.6967, and the determination coefficient of the regression equation is 0.8884, and the slope is 0.3372

, the standard deviation of the lowest detection concentration is 0.2554μA, and the detection limit is calculated to be 2.2 722 copies/μL; in each regression equation, I represents the current intensity, in μA; where ln(x) represents the target virus The concentration of the solution, the unit is copy number/μL; the slope refers to the slope of the regression line, the unit is

;The standard deviation of the lowest detection concentration refers to the standard deviation calculated from the current intensity values obtained from the results of three replicate experiments in a group with a target virus solution concentration of 0.005 copy number/μL, the unit is μA; where the detection Limit values are expressed in copies/μL. Figure 10 is a regression analysis chart drawn based on the experimental data in Table 5 and the results of the regression analysis for the groups using the wasabi peroxidase test and the intercalator test respectively according to the experimental data in Table 5; In Figure 10, the X-axis is the concentration of the target virus solution, the X-axis unit is the copy number/μL, the Y-axis is the measured current intensity, the Y-axis unit is μA, and according to the test method used, it is wasabi peroxidase test and embedding agent testing.

Experimental Example 11: Using the first electrochemical sensing set to test the wasabi peroxidase on the target extract, and using the second electrochemical sensing set to test the intercalating agent on the target extract.

Please refer to Table 6 and FIG. 11. In Experimental Example 11, the target extract was tested for wasabi peroxidase using the first electrochemical sensing kit. The steps are as described in “Method for testing wasabi peroxidase using the first electrochemical sensing kit”. The target biological substance sample used is a sample including the first target biological substance, the sample including the first target biological substance is the target extract, the specimen is serum, and the supernatant after the specimen is centrifuged is used as the diluent. The capture probe on the skeleton E is the first capture probe. In the step of "preparing each test sample by diluting the target extract solution sequentially, the target extract solution has a concentration of 0.005fM, 0.05fM, 0.5fM, 5fM, 50fM, 500fM, 5,000fM and 50,000fM as each test sample, wherein the detection probe is the first detection probe, wherein in "...... then, each of the washed skeletons E is respectively added with a redox enzyme diluted with a 0.1M phosphate buffer for reaction, In the step of "... then, the washed skeleton E is respectively added with the redox enzyme diluted with 0.1M phosphate buffer to react with the second marker on the redox enzyme to bind to the first marker on the detection probe...", the wasabi peroxidase labeled with streptavidin is diluted 25 times with 0.1M phosphate buffer, and the reaction time is 15 minutes, wherein the detection probe is the first detection probe; wherein in the step of "... then, the washed skeleton E is respectively added with the redox enzyme diluted with 0.1M phosphate buffer to react with the second marker on the redox enzyme to bind to the detection probe In the step of "the first marker ...", the wasabi peroxidase labeled with spheroidal avidin was diluted 25 times with 0.1M phosphate buffer, and the reaction time was 15 minutes; in addition, in Experimental Example 11, the target extract was tested for the embedding agent using the second electrochemical sensing kit, and the steps are referred to the "method for testing the embedding agent using the second electrochemical sensing kit", wherein the target biological substance sample used is a sample including the first target biological substance, and the sample including the first target biological substance is the target extract, The sample is serum, and the supernatant after centrifugation of each sample is taken as the diluent, wherein the capture probe on the skeleton E is the first capture probe, wherein in the step of "diluting the target biological substance sample with the diluent to complete the preparation of each test sample", the target extract is serially diluted so that the concentration of the target extract is 0.005fM, 0.05fM, 0.5fM, 5fM, 50fM, 500fM, 5,000fM and 50,000fM respectively, wherein the detection probe is the first detection probe; Experimental Example 1 The experimental results of 1 are shown in Table 6 and FIG. 11. Table 6 shows the current intensity measured at each concentration of the target extract when the specimen used is serum, wherein the concentration unit of the target extract is fM, wherein the unit of the current intensity is μA, and the test methods are divided into wasabi peroxidase test and intercalating agent test. In addition, according to the experimental data in Table 6, regression analysis is performed on the groups using the wasabi peroxidase test and the intercalating agent test, respectively. The regression equation of the regression line obtained by regression analysis is as follows, wherein the wasabi peroxidase test and the intercalating agent test are respectively used. The regression equation for sunflower peroxidase test is I=0.3249×ln(x)+1.9529, the coefficient of determination of the regression equation is 0.9653, the slope is 0.3249μA/fM, the standard deviation of the lowest detection concentration is 0.0416μA, and the detection limit is calculated to be 0.3841fM; the regression equation for intercalator test is I=0.8411×ln(x)+6.9862, the coefficient of determination of the regression equation is 0.9653, the slope is 0.3252μA/fM, and the lowest detection concentration is The standard deviation of the minimum detection concentration is 0.2183μA, and the detection limit is calculated to be 2.0138fM. In each regression equation, I represents the current intensity, and the unit is μA; ln(x) represents the concentration of the target extract, and the unit is fM; the slope refers to the slope of the regression line, and the unit is μA/fM; the standard deviation of the lowest detection concentration refers to the standard deviation calculated from the current intensity values obtained from the three repeated experimental results in the group with a target extract concentration of 0.005fM, and the unit is μA; the detection limit is fM. Figure 11 is a regression analysis chart drawn based on the experimental data in Table 6 and the results of regression analysis of the test methods used in Table 6, respectively, for the groups of the wasabi peroxidase test and the embedding agent test; in Figure 11, the X-axis is the concentration of the target extract, the X-axis unit is fM, the Y-axis is the measured current intensity, the Y-axis unit is μA, and the test methods used are the wasabi peroxidase test and the embedding agent test.

Experimental Example 12: Wasabi peroxidase test using the first electrochemical sensing kit on parathyroxine solution

，回歸方程式的決斷係數為0.9812，最低偵測濃度的標準差為0.12 μA，計算出偵測極限值為1.51pg/mL，於回歸方程式中，其中I代表電流強度，單位為μA；其中ln(x)代表副甲狀腺素溶液的濃度，單位為pg/mL；斜率指回歸 直線的斜率，單位為

其中最低偵測濃度的標準差指於副甲狀腺素溶液 的濃度為0.0001pg/mL的組別中，由三重複實驗結果獲得的電流強度值計算獲得的標準差，單位為μA；其中偵測極限值的單位為pg/mL。圖12為根據表7的實驗數據，及根據表7的實驗數據進行回歸分析的結果，所繪成的回歸分析圖；圖12中X軸為副甲狀腺素溶液的濃度，X軸單位為pg/mL，Y軸為測得的電流強度，Y軸單位為μA。 Please refer to Figure 12. In Experimental Example 12, the parathyroxine solution was tested using the first electrochemical sensing kit for wasabi peroxidase. For the steps, please refer to "Using the First Electrochemical Sensing Kit to Test Wasabi Peroxidase""Method", the target biological substance sample used is a sample including the second target biological substance, the sample including the second target biological substance is a parathyroxine solution, the sample is serum, and the supernatant after centrifugation of the sample is taken liquid as a diluent, in which the capture probe on the skeleton E is the third capture probe. In the step of "diluting the target biological substance sample with the diluent to complete the preparation of each detection sample", for the parathyroid The parathyroxine solution was serially diluted so that the concentrations of the parathyroxine solution were 0.0001pg/mL, 0.001pg/mL, 0.01pg/mL, 0.1pg/mL, 1pg/mL, 10pg/mL and 100pg/mL as each test sample. , wherein the detection probe is the second detection probe, in which "...then add oxidoreductase diluted with 0.1M phosphate buffer to each scaffold E after washing for reaction, so that The second label on the redox enzyme is bound to the first label on the detection probe..." In the step, wasabi labeled with streptavidin was treated with 0.1M phosphate buffer. The peroxidase was diluted 200 times, and the reaction time was 10 minutes; the experimental results of Experimental Example 12 are shown in Table 7 and Figure 12. Table 7 shows that the sample used is serum, and the parathyroxine solutions of various concentrations are respectively The current intensity measured by the wasabi peroxidase test, in which the concentration unit of parathyroxine solution is pg/mL, and the unit of current intensity is μA; in addition, regression analysis is performed based on the experimental data of the experiment in Table 7, and obtained through regression analysis The regression equation of the regression line is as follows. The regression equation when the specimen is saliva is expressed as: I=0.2371×ln(x)+9.3338, with a slope of 0.2 371

, the determination coefficient of the regression equation is 0.9812, the standard deviation of the lowest detection concentration is 0.12 μA, and the calculated detection limit is 1.51pg/mL. In the regression equation, I represents the current intensity in μA; where ln ( x) represents the concentration of parathyroxine solution, the unit is pg/mL; the slope refers to the slope of the regression line, the unit is

The standard deviation of the lowest detection concentration refers to the standard deviation calculated from the current intensity values obtained from the results of three replicate experiments in the group with a parathyroxine solution concentration of 0.0001pg/mL, in μA; where the detection limit Values are in pg/mL. Figure 12 is a regression analysis graph drawn based on the experimental data in Table 7 and the results of regression analysis based on the experimental data in Table 7; the X-axis in Figure 12 is the concentration of parathyroxine solution, and the X-axis unit is pg/ mL, the Y-axis is the measured current intensity, and the Y-axis unit is μA.

Experimental Example 13: Using the first electrochemical sensing kit to test the target protein solution for wasabi peroxidase

， 回歸方程式的決斷係數為0.9812，最低偵測濃度的標準差為0.11μA，計算出偵測極限值為0.33ng/mL，於回歸方程式中，其中I代表電流強度，單位為μA；其中ln(x)代表目標蛋白溶液的濃度，單位為ng/mL；斜率指回歸直線的斜率，單 位為

其中最低偵測濃度的標準差指於目標蛋白溶液的濃度為3.125ng/ mL的組別中，由三重複實驗結果獲得的電流強度值計算獲得的標準差，單位為μA；其中偵測極限值的單位為ng/mL。圖13為根據表8的實驗數據，及根據表8的實驗數據進行回歸分析的結果，所繪成的回歸分析圖；圖13中X軸為目標蛋白溶液的濃度，X軸單位為ng/mL，Y軸為測得的電流強度，Y軸單位為μA。 Please refer to Figure 13. In Experimental Example 13, the target protein solution was tested for wasabi peroxidase using the first electrochemical sensing kit. For the steps, please refer to "Method for testing wasabi peroxidase using the first electrochemical sensing kit." ”, the target biological substance sample used is a sample including the second target biological substance, the sample including the second target biological substance is the target protein solution, the sample is serum, and the supernatant after centrifugation of the sample is taken as Diluent, in which the capture probe on the skeleton E is the third capture probe, and in the step of "diluting the target biological substance sample with the diluent to complete the preparation of each detection sample", the target protein solution is Serial dilution, so that the concentrations of the target protein solution are 3.125ng/mL, 6.25ng/mL, 12.5ng/mL, 25ng/mL, 50ng/mL, 100ng/mL and 200ng/mL as each detection sample, in which the detection probe is the third detection probe, in which "...then add redox enzyme diluted with 0.1M phosphate buffer to each of the washed scaffolds E for reaction, so that the oxidoreductase on the redox enzyme In the step of binding the second label to the first label on the detection probe...", wasabi peroxidase labeled with streptavidin was subjected to 200 ℃ in 0.1M phosphate buffer. Double dilution, and the reaction time is 10 minutes; the experimental results of Experimental Example 12 are shown in Table 7 and Figure 12. Table 7 shows that the sample used is serum, and the wasabi peroxidase test was performed on the target protein solutions of various concentrations. The measured current intensity, in which the concentration unit of the target protein solution is ng/mL, and the unit of current intensity is μA; in addition, regression analysis is performed according to the experimental data of the experiment in Table 8, and the regression equation of the regression line obtained through regression analysis is as follows The regression equation in which the specimen is saliva is expressed as: I=0.9999×ln(x)+3.2009, with a slope of 0.9999

, the determination coefficient of the regression equation is 0.9812, the standard deviation of the lowest detection concentration is 0.11μA, and the calculated detection limit is 0.33ng/mL. In the regression equation, I represents the current intensity in μA; where ln ( x) represents the concentration of the target protein solution, the unit is ng/mL; the slope refers to the slope of the regression line, the unit is

The standard deviation of the lowest detection concentration refers to the standard deviation calculated from the current intensity values obtained from the results of three replicate experiments in the group with a target protein solution concentration of 3.125ng/mL, in μA; where the detection limit value The unit is ng/mL. Figure 13 is a regression analysis graph drawn based on the experimental data in Table 8 and the results of regression analysis based on the experimental data in Table 8; the X-axis in Figure 13 is the concentration of the target protein solution, and the X-axis unit is ng/mL , the Y-axis is the measured current intensity, and the Y-axis unit is μA.

Experimental Example 14: Using the first electrochemical sensing kit to test the wasabi peroxidase on the microRNA solution, and using the second electrochemical sensing kit to test the intercalating agent on the target extraction solution

Please refer to Table 9 and Figure 14. In Experimental Example 14, the microRNA solution was tested using the second electrochemical sensing kit for the intercalating agent. The steps were the same as "Method for testing the intercalating agent using the second electrochemical sensing kit." Basically the same, the target biological substance sample used is a sample including the fourth target biological substance, the sample including the fourth target biological substance is a microRNA solution, the sample is serum, and the supernatant of each sample after centrifugation is taken The supernatant is used as a diluent, and the capture probe on the skeleton E is the fourth capture probe. In the step of "diluting the target biological substance sample with the diluent to complete the preparation of each detection sample," The ribonucleic acid solution was serially diluted so that the concentrations of the microRNA solution were 5fM, 50fM, 500fM and 5,000fM respectively; the difference between Experimental Example 14 and "Method of using the second electrochemical sensing kit for intercalator testing" is that ".. ....Then add 20 μL of 1 mg/mL framework E solution to 10 μL of each detection sample, and add the detection probe aqueous solution and intercalating agent, then shake and mix with a oscillator for 10 minutes to make the The target biological substance is captured by the capture probe on the skeleton E, and the detection probe is combined with the target biological substance captured by the capture probe on the skeleton E, and the intercalating agent embeds the hybridization of the capture probe and the target biological substance. In the step "...and after adding the detection probe aqueous solution and the intercalating agent..." in the step "..." Only the intercalating agent is added, and the intercalating agent added is 10 μL of 2mM neutral red; the experimental results of Experimental Example 14 are shown in Table 9 and Figure 14. Table 9 shows the results at various concentrations when the specimen used is serum. The current intensity measured by the microRNA solution, where the concentration unit of the microRNA solution is fM, and the unit of current intensity is μA. In addition, regression analysis is performed based on the experimental data in Table 9, and the regression equation of the regression line is obtained through regression analysis. As explained below, the regression equation is I =1.6875×ln(x)+6.698, the determination coefficient of the regression equation is 0.984, the slope is 1.6875μA/fM, the standard deviation of the lowest detection concentration is 0.31μA, and the calculated detection limit value is 0.55fM; where I represents the current Intensity, in μA; where ln(x) represents the concentration of the target extraction solution, in fM; the slope refers to the slope of the regression line, in μA/fM; where the standard deviation of the lowest detection concentration refers to the concentration of the target extraction solution In the 5fM group, the standard deviation is calculated from the current intensity values obtained from the results of three repeated experiments, in μA; the unit of the detection limit value is fM. Figure 14 is a regression analysis graph drawn based on the experimental data in Table 9 and the results of regression analysis based on the experimental data in Table 9; in Figure 11, the X-axis is the concentration of the target extraction solution, and the X-axis unit is fM, Y The axis is the measured current intensity, and the Y-axis unit is μA.

In summary, the present invention provides a modified magnetic iron metal organic framework, a preparation method thereof and an electrochemical sensing kit including a modified magnetic iron metal organic framework, and has been confirmed by various examples to have modified magnetic iron. The analysis of target biological substances by combining metal organic frameworks with electrochemical sensors not only overcomes the problem of insufficient sensitivity of general metal organic frameworks combined with electrochemical sensors, but also has good specificity and a wide quantitative range, making it a good choice for metals. Contributions to the field of organic frameworks combined with electrochemical sensors for analysis of biological substances.

A0101_OR_PSEQ.xml

Claims (10)

A modified magnetic iron metal organic framework, including: a magnetic iron metal organic framework, a chemical modification and a capture probe, wherein the magnetic iron metal organic framework is composed of ferric ions, 2-methylimidazole and L -A metal-organic framework formed by coordination and combination of histidine acid, and the magnetic ferro-metal organic framework includes an accommodation space in which a magnetic nanoparticle, the chemical modification and the capture probe are accommodated Covalently connected to the surface of the metal organic framework; wherein the chemical modification is thionine; wherein the capture probe is used to bind to a target biological substance in a sample; wherein the capture probe is an oligonucleotide, Antibodies or peptide chains. The modified magnetic iron-metal organic framework as described in claim 1, wherein the target biological substance is a nucleic acid, a peptide, a protein or a compound. The modified magnetic iron-metal organic framework as described in claim 1, wherein the magnetic iron-metal organic framework is composed of magnetic nanoparticles, L-histidine acid, polyvinylpyrrolidone, water and methanol, and then chlorine is added Iron hexahydrate and water are then left to react at 4°C. Finally, 2-methylimidazole and methanol are added and mixed at room temperature to react. The modified magnetic ferrometal-organic framework as described in claim 3, wherein the particle size of the magnetic nanoparticles is less than 1,000 nanometers. The modified magnetic iron metal organic framework as described in claim 1, wherein the sample is saliva, urine, serum, plasma, whole blood, cells, cell extracts, lymph, cerebrospinal fluid, amniotic fluid, alveolar wash fluid, sweat, tears, sputum, pleural effusion, ascites, joint fluid, semen or feces. A method for preparing a modified magnetic iron metal organic framework as described in claim 1, the steps include adding 1-ethyl-3(3-dimethylaminopropyl)carbodioxide and N-hydroxysuccinidine Amine, magnetic iron metal organic framework and water are mixed and reacted at room temperature, and then rinsed with pure water. Then thionine, capture probe and water are added and mixed and reacted at room temperature to obtain a modified metal organic framework. The preparation method as described in claim 6, further comprising: after completing the addition of thionine, capture probe and water, mixing the reaction at room temperature, first rinsing with pure water, and then drying in an oven to obtain modified of metal-organic frameworks. An electrochemical sensing kit includes: an electrochemical sensor, including a working electrode, the working electrode includes a surface for detecting current changes, and a back surface opposite to the surface, the working electrode A magnet is provided on the back; a modified magnetic ferrometal-organic framework as described in claim 1; a detection probe for binding to the target biological substance, and a first marker is modified on the detection probe; and An oxidation-reduction enzyme is used to perform an oxidation-reduction reaction, and the oxidation-reduction enzyme is modified with a second label, and the second label is used to bind to the first label. An electrochemical sensing kit includes: an electrochemical sensor for performing an electrochemical analysis, the electrochemical sensor includes a working electrode, and the working electrode includes a surface for detecting current changes, And a back side opposite to the surface, the back side of the working electrode is provided with a magnet; a modified magnetic iron metal organic framework as described in claim 1; and an embedding agent for embedding the capture probe and the The target biological substance binds and undergoes an oxidation-reduction reaction; the capture probe is an oligonucleotide; and the target biological substance is a nucleic acid. The electrochemical sensing kit according to claim 9, further comprising a detection probe, the detection probe is used to combine with the target biological substance, and the intercalating agent will be embedded in the detection probe and the target biological substance. The binding site, wherein the detection probe is an oligonucleotide.